Discovery of a Novel Variant of SEMA3A in a Chinese Patient with Isolated Hypogonadotropic Hypogonadism

Semaphorin (SEMA) has an important role in nerve development, organ formation, immune response, angiogenesis, and tumor growth. SEMA can regulate the growth and branching of axons, the morphology of dendrites, and the migration of neurons. The loss-of-function in SEMA and its receptors PLXNs and NRP affect the migration of GnRH neurons, leading to idiopathic hypogonadotropic hypogonadism (IHH). As a member of the SEMA family, SEMA3A has an important role in axonal rejection, dendritic branching, synaptic formation, and neuronal migration. There are more and more SEMA3A variants identified in IHH patients. In this study, we identified a novel SEMA3A variant (c.1369A > G (p.T457A)) in a male nIHH patient. Functional studies indicated that the T457A SEMA3A variant led to the defect of FAK phosphorylation and GN11 cell migration, which strongly argued in favor of its pathogenic effect in the nIHH patient. Our findings substantiated that the 435–457 position of SEMA3A might be very important for the secretion of SEMA3A. Haploin-sufficiency of SEMA3A in humans was sufficient to cause the IHH phenotype. SEMA3A variants might have a role in modifying the IHH phenotype, according to the variants at different positions of SEMA3A. SEMAs and its receptors formed a complex network, and other members of the SEMA-signaling pathway might also be involved in the pathogenesis of IHH.


Introduction
SEMA is a family of proteins widely involved in various physiological and pathological processes, such as nerve development, organ formation, immune response, angiogenesis, and tumor growth. According to its structural characteristics, SEMA family can be divided into 8 subtypes [1]. e specific receptors of SEMA family mainly include plexin (PLXN) and neuropilin (NRP). During the development of the nervous system, SEMA can regulate the growth and branching of axons, the morphology of dendrites, and the migration of neurons. Recent studies have found that some mutations in SEMA and its receptors PLXNs and NRP affect the migration of GnRH neurons, resulting in delayed puberty maturation or infertility [2], leading to idiopathic hypogonadotropic hypogonadism (IHH).
In this study, we identified a novel SEMA3A variant in a large cohort of IHH patients from China. Functional studies suggested that the SEMA3A mutation led to the defect of FAK phosphorylation and GN11 cell migration.

Whole Exome Sequencing.
e exome depth algorithm was used for analyzing copy number variation (CNV) [9]. e detected variants were verified by PCR-Sanger sequencing, and the sequences of PCR primers are given in Table S1. Cosegregation analyses were conducted on all family members if available. e pathogenicity prediction of the identified variants is described previously [3].
e Genome Aggregation Consortium (gnomAD) and the Chinese control individuals were used to determine the allelic frequencies for each variant. In addition, we used InterVar (http://wintervar. wglab.org/) to determine variant classification based on American College of Medical Genetics and Genomics (ACMG) guidelines [10].

Plasmids and Cell Transfection.
e pRK5-SEMA3A-Flag constructs (SEMA3A cDNA, GenBank accession no. NM_006080) were constructed at Youbao Biological Company. We used the QuikChange mutagenesis kit (Stratagene, La Jolla, CA) to make variants in the pRK5-SEMA3A-Flag constructs by site-directed mutagenesis. All constructs were verified by nucleotide sequencing.
Plasmid transfections were performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) reagents according to the manufacturer's protocol.

Expression and Secretion of SEMA3A Protein.
We transfected pRK5-SEMA3A-Flag and T457A mutant plasmid in the HEK293T cells through Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA). We used the Ultra 3K filter tubes (30 kDa NMWL, Millipore) to concentrate the media of the HEK293T cells. Western blot was used to detect the expression and secretion of SEMA3A protein. Each experiment was performed at least three times.

Detection of FAK Phosphorylation in GN11 Cells.
GN11 cells maintained in the serum-free medium were stimulated with WT and mutant SEMA3A concentrated conditioned media for 20 minutes. Western blot was used to detect the expression of pFAK and FAK in cell lysates. Each experiment was performed at least three times.

Transwell
Assay. GN11 cells (1 × 10 4 cells) planked into transwell chambers with 8 μm pore filters (Sigma, WA) were maintained in SEMA3A and its mutant conditioned media overnight. e cells removed mechanically from the upper side of the filters were stained by DAPI and counted through the fluorescence microscope.

Statistical
Analysis. Statistical analysis was performed by the Prism 6.01 (GraphPad Software, San Diego, CA) with the unpaired two-tail Student's t-test or a repeated-measure ANOVA followed by Bonferroni post hoc tests to analyze the data for differences. P < 0.05 was considered significant. e distribution of data points is shown as mean ± S.E.M.

Identification of a Novel SEMA3A Variant in Patients with IHH.
We screened a SEMA3A heterozygous variant (c.1369A > G (p.T457A)) in a male proband of nIHH ( Figure 1(b)). e T457A variant was located in the SEMA domain ( Figure 1(c)).

Functional Analysis of Novel SEMA3A Variant.
rough Western blot analysis, both wild-type (WT) SEMA3A protein and T457A mutant protein were expressed in HEK293T cells. But, the T457A mutant protein was defective in secretion. en, we added the conditioned medium containing concentrated WT and T457A mutant SEMA3A protein to GN11 cells and detected the phosphorylation level of FAK (pFAK) in GN11 cells by Western blot. We found that pFAK in GN11 cells stimulated by WT SEMA3A protein was significantly increased compared with that in the negative control, but the T457A mutant protein could not effectively induce FAK phosphorylation in GN11 cells (Figure 2(b)).
Furthermore, we detected the migration of GN11 cells maintained in WT SEMA3A and T457A mutant protein by the transwell assay. Compared with WT SEMA3A protein, the migration of GN11 cells was significantly decreased under the stimulation of T457A mutant protein (Figure 2(c)). It suggested that the T475A variant resulted in the loss of SEMA3A function.

Discussion
e development of the mammalian reproductive system depends on GnRH neurons located in the hypothalamus.
GnRH neurons first appear in the medial olfactory placode and migrate to the cribriform plate and forebrain along the axons of olfactory vomeronasal neurons. en, GnRH neurons enter the forebrain, migrate to the developing olfactory bulb, and finally reach the ventral hypothalamus. As a neuroendocrine cell, they release GnRH into the pituitary portal circulation [11] and regulate the "hypothalamus pituitary gonad (HPG) axis" of human beings. Accordingly, loss-of-function for molecules, such as SEMA3A that control olfactory or vomeronasal axon-patterning perturbs GnRH neuron migration, leads to delayed or absence of pubertal maturation and infertility [2]. e normal secretion of SEMA3A plays an important role in the normal function of SEMA3A.
In this study, we further investigated 196 Chinese IHH patients and found a novel SEMA3A variant (p.T457A) in a male nIHH patient. Functional study suggested that it led to loss-of-function in SEMA3A, and it resulted in impaired secretion of SEMA3A. Dai et al. found three SEMA3A variants (R197Q, R617Q, and V458I) in 177 Chinese IHH patients by whole exome sequencing. Functional study suggested that all three variants caused loss-of-function in SEMA3A [3], and the V458I variant led to impaired secretion of SEMA3A. Hanchate et al. identified a frameshifting small deletion (D538fsX31) and several heterozygous missense variants (D538fsX31, R66W, V435I, N153S, I400V, T688A, and R733H) in SEMA3A in KS patients. Among them, three variants resulted in impaired secretion of SEMA3A (D538fsX31, R66W, and V435I) [4]. In conclusion, the R66W, N153S, R197Q, I400V, V435I, p.T457A, and V458I variants are located in SEMA domain, all in heterozygosity, but the R66W, V435I, V458I, and p.T457A variants caused impaired secretion of SEMA3A (Figure 3). It suggested the mutations at positions 435-457 in the SEMA region are likely to lead to impaired secretion of SEMA3A, and the 435-457 position is very important for the secretion of SEMA3A. Haploin-sufficiency of SEMA3A in humans was sufficient to cause the IHH phenotype [3,4,6,12].
SEMA3A was considered to play an important role in axonal rejection, dendritic branching, synaptic formation,    and neuronal migration [8]. SEMA3A knock-out mice exhibited abnormal olfactory bulb innervation and altered development of GnRH neurons [7]. Hanchate [12]. It seems possible that SEMA3A variants might modify the IHH phenotype, according to the variants at different positions of SEMA3A. Furthermore, SEMAs and its receptors form a complex network. Some SEMAs can bind to a variety of receptors. On the other hand, some SEMA receptors can bind to many kinds of SEMAs [13]. In this study, we found the proband (II : 3) carrying T457A SEMA3A, R415S SEMA4G, G421E PLXNA4, A1891T PLXND1, and S134D FGFR1 variants. Giacobini et al. reported that SEMA4D and plexin B1 are important for the guidance of migrating GnRH neurons [14]. It suggested that other members of the SEMA-signaling pathway might also be involved in the pathogenesis of IHH.  [3]. In this study, the proband with the SEMA3A variant (T457A) also carry p.S134D variant in FGFR1. It suggested that the SEMA3A-NRP1/NRP2-PLXNA1 signaling pathway might cooperate with the PROK2-PROKR2 or FGF8-FGFR1 signaling pathway in the pathogenesis of IHH.
In summary, we present a new SEMA3A variant identified in 196 Chinese IHH patients. From our results, we concluded that the p.T457A variant affected the secretion of SEMA3A and, furthermore, influenced signaling activity of SEMA3A, which led to its pathogenic effect in the nIHH patient. Variants at positions 435-457 in the SEMA region was likely to lead to impaired secretion of SEMA3A, and the 435-457 position might be very important for the secretion of SEMA3A. Furthermore, SEMAs and its receptors form a complex network, and other members of the SEMA-signaling pathway might also be involved in the pathogenesis of IHH. Otherwise, it will be worthful to study whether the SEMA3A-NRP1/NRP2-PLXNA1 signaling pathway cooperated with the PROK2-PROKR2 or FGF8-FGFR1 signaling pathway in the pathogenesis of IHH.

Data Availability
e data generated during the study are available from the corresponding author upon request.